evolution of alternate modes of development in ascidians

lEvolution of Alternate Modesof Development in Ascidians

William R. Jeffery and Billie J. Swalla

SummaryAscidians have evolved alternate modes of developmentin which the conventional tadpole larva is remodeled oreliminated. Adultation, the precocious development ofadult features in the larval head, is caused bysuperimposing the larval and adult differentiationprograms. Caudalization, the addition of muscle cells tothe larval tail, is caused by enhancing muscle inductionor increasing the number of muscle cell divisions beforeterminal differentiation. Adultation and caudalizationare correlated with increased egg size, suggestingdependence on maternal processes. Anural develop-ment, the elimination of the larval stage, is caused bymaternal and zygotic events resulting in abbreviationand deletion of larval developmental programs. Anexample of a maternal change in anural species is themodification of the egg cytoskeleton during oogenesis,whereas a zygotic change may involve altered cellinteractions during embryogenesis. Interspecific hybrid-ization experiments suggest that some aspects of anuraldevelopment may be caused by loss-of-function mu-tations. The dissociation of developmental programs is akey process in changing the mode of development inascidians.

lntroductionHow have different modes of development arisenduring evolution? Differences in ontogeny are tra-ditionally explained by heterochrony, a shift in therelative timing of $ifferent developmental processesduring evolution('-''. Although heterochrony is welldocumented and considered to be the most commonmeans of generating morphological changes in develop-mentt'''), it is difficult to explain how novel develop-mental features arise and existing features are deletedby this process. An additional complication is thatalthough some heterochronic events are based on non-tempo.ral changes that occur much earlier in develop-ment(*), early developmental events are believed to beconstrained and resistant to evolutionary change(s). Asearly as 1896, however, F. R. Lillie("' showed that thedevelopment of specialized larval shell muscles in theclam Unio is mediated by a change in the rate andpattern of spiral cleavage. Therefore, early develop-mental processes may show more evolutionary plas-

ticity than has been appreciated. Despite recentprogress in understanding the basis of embryonicpattern formation, little is known about the mechan-isms underlying evolutionary change in development.

The life cycles of many invertebrate phyla contairrlarval and adult phases. In some of these groups, theconventional larva has been remodeled by adding newstructures or eliminating the larval phase, resulting indirect developmenl(7). when radicaf differences inlhemode of development exist in congeneric species, theyhave served as model systems for studying evolutionarychanges in developmental processes(o). In this essay, wedescribe alternate modes of development in ascidiansand review how variations in conventional developmentmay contribute to understanding mechanisms ofontogenetic change during evolution.

'Asc id ians

Ascidians are lower chordates (Subphylum Urochor-data, Class Ascidiacea) with a life cycle containing bothlarval and adult phases. The adult is sessile andenclosed in a sac-like tunic. The major adult organsinclude a digestive tube with terminal siphons, abranchial basket with gill slits, a circulatory systempowered by a tubular heart, and a muscular mantle.Ascidians can be solitary or colonial, and many speciesreproduce by asexual (budding) as well as sexualprocesses.

Although adult ascidians bear little superficialresemblence to other chordates, their tadpole larvaehave distinct chordate features, including a notochordand dorsal nervous system. The tadpole head contains abrain equipped with two pigmented sensory organs (theocellus and otolith), mesenchyme cells, and endoderm,whereas the tail consists of a notochord, flanked by adorsal spinal cord, a ventral strand of endoderm, andlateral bands of striated muscle cells (see Figs 2A and3). The surface of the larva is covered by epidermis,which secretes a thin larval tunic during embryogenesis.

After hatching, the tadpole swims for up to severaldays, then attaches to a substrate and undergoesmetamorphosis. There is no feeding in the larval phase,which functions exclusively for dispersal. Duringmetamorphosis, the tail is retracted into the head, thelarval tissues are resorbed, and adult structuresdifferentiate from rudiments in the head.

Embryonic DevelopmentAn advantage of using ascidians for evolutionarystudies is that their development is well known. The celllineage of ascidian embryos haq _been investigated indetail by direct observations(b'e) and cell tracingmethods(10-12). These studies have shown that ascidiancell lineages are qualitatively invariant, both within andbetween species. Figure L shows the fate map of the 8-cell embyro. Cell fates are the same on each side of theplane of bilateral symmetry, except for the otolith and

BioEssays Vol. 14, No. 4 - April 1992 219

Ep ide rm isSecondary Muscle

Primary MuscleNotochordEndodsrmMesenchyme

Fig. 1. Fate map of the 8-cell ascidian embryo showing theorigin of larval tissues. The bilaterally symmetric embryo isviewed from the side with the animal pole on the top and theanterior pole on the right. Lineages in italics are known to beaffected during anural development.

ocellus, which differentiate unilaterally in the brain(13).The anterior-animal (a4.2) quadrant gives rise to theepidermis, brain, and brain sensory organs; theposterior-animal (b4.2) quadrant to epidermis and tailmuscle; the anterior-vegetal (A4.1) quadrant to noto-chord, endoderm, spinal cord, and tail muscle; and theposterior-vegetal (B4.1) quadrant to notochord, endo-derm, mesenchyme, and tail muscle. Therefore,epidermis, endoderm, notochord, and tail muscle havemultiple blastomere origins in ascidian embryos.

Two types of larval muscle cells are distinguished bytheir lineage, method of determination, and finallocation in the tail. The primary muscle cells, whichoriginate exclusively from the B4.1 quadrant, producethe anterior and middle portions of the tail muscle,whereas the secondary muscle cells, which are derivedfrom the b4.2 and A4.1 quadrants, produce theposterior portion of the tail muscle("r. The embryoniccells destined for larval structures are restricted in fateby the 110-cell stage(12), undergo a few more divisions,then cease dividing and differentiate. In contrast,embryonic cells destined to form adult structures(primarily endoderm and mesenchyme) continue todlvide during the larval phase and differentiate in thejuvenile after metamorphosis('"'.

Ascidians show both cell autonomous and inductivemethods of cell determination. The primary musclecells are specified autonomously by ooplasmic determi-nants. Although the muscle determinants have not beenidentified, thely probably reside in the myoptut-(1s), aspecialized ooplasm segr^egated to the primary musclelineaee during cleavagstur. There is evidence that theendoierm(16)Iepiderrial(17), and notochord(l8) lineagesare also determined by cell autonomous processes. Incontrast the larval brain, sensory organs, and secondarymuscle cells are determined conditionally by inductivecell interactionr(13'1e-22).

During gastrulation, which is initiated by .i{rvagi-nation of endoderm cells at the vegetal pole(o', thepresumptive notochord, mesenchyme and muscle cellsmove over the lips of the blastopore and the ectoderm

spreads over the surface of the embryo. Subsequently,the ectoderm elaborates a neural tube which later formsthe larval brain. Once inside the embryo, the endodermcells migrate anteriorly into the future larval head andthe noto=chord cells intercalate at the midline, swell, andextend posteriorly to form the tail(23). During thetailbud stage, the otolith and ocellus appear in thebrain, and bands of muscle cells differentiate in the tail.

Adultation and Caudalization: Adding NewDimenisions to the Tadpole Larva

Ascidians have evolved alternate modes of develop-ment in which the larval phase has been remodeled indifferent ways (Fig. 2). These modes of developmentwere first stuaieOln detail by N. J. Berrill(r4'24'27'28),whose original studies provide an important foundationfor this essay.

Adultation is a mode of development in which adultstructures differentiate precociously in the tadpolehead. This developmental alternative reduces the

' @A

d l ( @

V

Fig. 2. Diagrams showing the stucture of the tadpole larva andalternate developmental modes in ascidians. (A) Conven-tional tadpole of Molgula oculata. (B) Hatched anuralembryo of Molgula occulta. (C) Tadpole of Molgula citrinawith minimal adultation. (D) Tadpole of Botryllus grgas withintermediate adultation. (E) Tadpole of Ecteinascidia turbi-nata with extreme adultation. The row of cells in the tails ofthe tadpole larvae shown in A and C-E represent thenotochord. The structures present in the heads of tadpolelarvae shown in A and C-E represent brain sensory organs(fltled spheres; otolith in A and C, otolith and ocellus in D andE) or precociously developed adult structures in C-E. Thestructure in the hatched anural embryo (A) represents thebrain. Data from Berrill(ia) and Swalli and Jeffery(3s)'

EpidermisBrainBrain Sensory Organ(s)

Sacondary MuscleNolochotdEndodermSpinal Cord

220 BioEssays Vol. 14, No. 4 - April 1992

l'--

Table 1. Egg sizes and temporal relationships in ascidians with conventional development, anural development, andadultation

Species(Mode)

Egg diameter(pm)

Difference inegg volume

Time until Difference inhatching (hr) hatching time

Molgula oculata 80(Urodele conventional)

Molgula occulta 100(Anural)

Molgula citrina 210(Urodele with adultation)

Botryllus gigas 450(Urodele with adultation)

Ecteinascidia turbinata 720(Urodele with adultation)

Data from Berrill(28) and Swalla and Jeffery(35)

I 1

Differences in egg volume and hatching time are shown relative to M. oculata.

T2

1.8X

12.7X

r82.0x

702.5X

150

190

420

0.9x

12.5X

17.3X

38.2X

interval between larval hatching and the beginning offeeding after metamorphosis. Adultation can beminimal (Fig. 2C), with early appearance of one or bothsiphons, a partial digestive tract, a few gill slits, and arudimentary heart, or it may involve more extensivedevelopment of adult structures (Fig. 2D, E). Inextreme cases, a miniature juvenile with a completedigestive tract, a branchial basket with numerous gillslits, a beating heart, and sometimes even a pre-formedasexual bud, develops within the larval head (Fig. 2E).Even in extreme adultation, however, the siphons donot open through the larval tunic, and feeding isdelayed until after settlement and metamorphosis. It issometimes speculated that extreme adultation led toneotony and the origin of the larvaceans, a class ofurochordates whose adult orsanization is similar to thatof the ascidian tadpole(2a).

-

Changes in the timing of gene expression accompanymorphological alterations in species with adultation.The ascidian genome contains a family of alpha actingenes, some of which are expressed^during embryonicdevelopment and others in the adult("). An alpha-actingene that is expressed in adult muscle cells of Styelaplicata, which forms a conventional tadpole, begins tobe expressed during the.lgrval phase in Molgula citina,which shows adultation('o'. The precocious expressionof the adult alpha-actin gene is confined to a class oflarval mesenghfme cells which may be the precursors ofadult muscle('"'.

Adultation is caused by superimposing the programsoflarval and adult differentiation. The crucial heteroch-ronic event appears to be retardation of larvaldevelopment, although acceleration of adult develop-ment also occurs in some species('"/. All ascidians withadultation are ovoviviparous and have relatively largeeggs (Table 1). Egg size (or yolk content) and the rateof larval development are inversely related in as-cidians(la), in part explaining why adultation increasesin species with larger eggs (Fig. 2C-E;Table 1). Thus,temporal changes that produce adultation in theswimming larva may be controlled by events that occur

170 320 720

Fig. 3. Caudalization of the tadpole larva. The top rowindicates the number of tail muscle cells in the tadpole, themiddle row cross-sections through the tadpole tail, and thebottom row the egg diameter in (A) Ciona intestinalis, (B)Halocynthia roretzi, (C) Stolonica socialis, and (D)..^ctein-ascidia turbinata. Data from Nishida and Satoh("' andBeriill04'24'27). Drawn in part after Berrill(27).

much earlier in development, such as the extent of yolkdeposition during oogenesis.

Caudalization is a mode of development in whichmuscle cells are added to the tadoole tail withoutchanging the number of other larval

^cells(1+'z+'n). 'I.6i,

alteration boosts the swimming ability of the larva andenhances dispersal. Similar to adultation, caudalizationcan be minimal or extreme (Fig. 3). The tadpole ofHalocynthia roretzi, which has 42 (rather than theconventional 36 to 38) tail muscle cells, is an example ofminimal caudalization (Fig. 3B). In this species, musclecells are added to the posterior tip of the tail.by theinduction of more secondary muscle cells("'"/. Ex-treme caudalization occurs in tadpoles of Stolonicasocialis, which have 378 tarl muscle cells, andEcteinascidia turbinata, which have 1J.43 tail muscle


cA

36

D

1134 Tai lMuscle Cel ls

Spinal cord

Notochord

Muscle cellbands

Endodermal strand

42 378

. l r A@ @@wx7ZZO Eggdiameter

cells(14). In these species, additional muscle cells areproduced by extra cell divisions before terminaldifferentiation, and the muscle cells are packed into therobust tail by increasing the number of muscle bands(Fig. 3C, D). Whether the number of cell divisions isincreased in the primary lineage, the secondary lineage,or both muscle lineages is unknown for species withextreme caudalization.

As shown in Figure 3, the extent of caudalization iscorrelated with egg size (some ascidians with very largeeggs show both adultation and extreme caudalization),suggesting that maternal events also control this modeof development. An intriguing possibility is thatavailability of stored cell cycle components limits theextent of larval muscle cell division during conventionaldevelopment and that the large eggs of species withextreme caudalization contain an expanded pool ofthese substances, which is segregated into the musclelineage during cleavage.

Anural Development: Erasing the Tadpole Larva

Elimination of the larval stage from the ascidian lifecycle is known as anural (or tailless) developmentrelative to conventional urod-ele (or tailed) develop-ment (Figs 2B and 4A)Q8'2e). Species with anurildevelopment are restricted to 2 of the 14 families ofascidians: the Styelidae and Molgnli6u"(zs-3r). Anuralspecies are probably derived from urodele ancestorsbecause species with urodele development predominatein the Styelidae and Molgulidae, and anural embryosshow morphological and biochemical vestiges ofurodele development (see below).

An important question is whether anural develop-ment evolved more than once in ascidians because theanswer provides insight into the number of geneticchanges that may be involved in the developmentalswitching mechanism. Anural development is likely tohave evolved independently in the Styelidae andMolgulidae because each of these families are moreclosely related to the Pyuridae, a family containing onlyurodele species, than they are to each other. Based ondifferences in embryonic and adult morphology, it hasalso been suggested that anural development arosemultiple times within the Molgulidaevo'"). The inde-pendent origin of anural development within the samefamily of ascidians suggests that this developmentalalternative may be mediated by a relatively smallnumber of genetic changes.

Anural ascidians inhabit uniform environments suchas subtidal sand or mud flats and shorelines withvigorous wave action. In these environments, watercurrents can be used for dispersal, and the selectiveadvantage for a swimming larva may be minimal. As inother cases of regressive evolution (e.g. loss ofpigmentation and eyisight in subterrurr"un uii-uls)(::),the selective value of losing a morphological feature is

Fig. 4. Hybridization between an anural and urodele ascidian.A. Hatched M. occulta embyro with no brain pigment cell ortail. B. Hatched M. occulta x M. oculata hybrid embryo witha brain pigment cell and a short tail. C. M. oculata tadpolewith a brain pigment cell (in an ot-olith) and a tail. Scale bar:20 pm. From Swalla and Jeffery(").

difficult to explain. A potential selective agent may beincreased metabolic economy: a mutation that reducesor eliminates a presumably useless structure, such asthe tail or otolith of anural embryos, would have aselective advantage because it saves energy formetamorphosis. The actual situation is probably morecomplicated, however, because anural and urodelespecies can be sympatric. For instance, the anuralascidian Molgula occulta and the urodele ascidianMolgula oculata inhabit the same sand flats off thenorthwest coast of Europe(28'34'3s). However, the size ofthe M. occulta population greatly exceeds that of M.

A

B

c

222 BioEssays Vol. '14, No. 4 - April 1992

t-oculataQs), suggesting that the anural species is a bettercompetitor in this habitat. The anural species alsoprobably reaches reproductive maturity earlier than theurodele congener (Swalla and Jeffery, unpublished).Hence, selective pressure to retain a swimming larvamay be maintained for dispersal of M. oculata tadpolesfrom parts of the sand flat that are already occupied bydeveloping M. occulta juveniles. Alternatively, M.oculata tadpoles are relatively weak swimmers, and thisspecies may be in the process of eliminating the larvalstage.

Dissociation, Abbreviation and Elimination ofDevelopmental Programs in Anural AscidiansThe dissociation, abbreviation, and elimination oflarval developmental programs is a fundamentalproperty of anural development. The pattern of earlycleavages is the same in anural and urodele embryos butdifferences in cell lineages appear after cleavage, andmorphogenetic processes are altered during or aftersastrulation that lead to the formation of a tailless6mbryo(28'2s '32'34'3s-4r) . The cell lineages that arechanged during anural development are shown inFigure 1.

A tail fails to materialize during anural developmentbecause there is no intercalatio.-n,-_.swelling, andextension of the notochord cells(28'3s). Although anotochord lineage is formed during cleavage, furtherdevelopment is arrested after gastrulation and thenotochord cells accumululgqg aplacode in the posteriorregion of anural Bmbryostrz'r)-ro'. In the anural ascidianM. occulta,, the notochord placode contains only 10 to20 cells(3s), a significanC reduction from the 40notochord cells that differentiate in the urodele larvaltail. The decrease in notochord lineage cells could becaused by a change in cell fate or a reduction in thenumber of cell divisions in the notochord lineage.Abbreviation, rather than elimination, of the noto-chord program has probably occurred because pre-sumptive notochord cells may induce the larvalbrai;(1e'20). which is retained in anural em-bryos(28-:z':s-37). Thus, retention of the notochordlineage in anural species may represent a developmen-tal constraint imposed by its inductive function duringembryogenesis.

Although there is no muscle cell differentiationduring anural development, a primary muscle lineage isformed during cleavage, and a significant number ofundifferentiated muscle cellrs- appear in the posteriorregion of anural €rnbryos(r)-r". According to dataobtained from northern blot and in situ hybridizationwith alpha actin and myosin heavy chain probes and invitro translation of embryonic mRNA, the musclelineage cells do not.d-etectably express genes encodingcontracti le proteins("). ln some anural species, how-ever, these, cells produce acetylcholinesterase(AChE)(35'37-3e), an eniyme expressed in the muscle

lineage of urodele embryos(42). These results show thaturodele features expressed in the same lineage can bedissociated during anural development.

AChE expression was used to quantify the musclelineage in M. occulta embryos("). Although cellnumbers vary between individuals, reflecting lack ofselection for a particular cell number, these embryosdevelop a mean of 20 AChE-positive muscle lineagecells, many fewer than the 36 to 38 muscle cells presentin urodele ascidians. Cleavage arrest experiments,which can be used to determine the lineage identity ofblastomeres expressing tissue-specific markers in as-cidians("". show that the AChE-positive cells are partof the primary muscle lineaget'>'"'t. In ascidians withconventional urodele develooment. 28 of the tailmuscle cells originate from the primary lineage,

- whereas the remainine 8 to 10 muscle cells arise fromthe secondary lineagelll), indicating that some of theprimary muscle cells and all of the secondary musclecells are deleted during anural development. Thefunctional significance of eliminating one musclelineage while abbreviating another is uncertain. How-ever, abbreviation could be a constraint related to anunknown inductive role of primary muscle cells duringascidian development.

The tail is not the only larval feature that is absent inanural ascidians. In contrast to urodele species (Fig.2A, C-E), anural embqyos lack brain sensory organs(Figs 28 and 4A)(28-3/'34-4r\, which are thoughi tocoordinate swimming and settling of the tadpole larva.An important exception is the anural molgulidBostrichobranchus digonas, which^.develops a brainpigment cell during embryogenesist"/. The pigment cellis not incorporated into a brain sensory organ, however,showing that the program of sensory organ differen-tiation is abbreviated in B. digonas embryos. Thus,depending on the anural species, sensory cell develop-ment is either abbreviated or eliminated. The develop-ment of a brain pigment cell in B. digonas also showsthat different aspects of neural differentiation (e. g.pigment cell and brain differentiation) can be dis-sociated from each other and from other urodelefeatures during anural development.

Anural development probably evolved in severaldiscrete steps. The first step must have been dis-sociation of larval developmental programs, whichwould be required for differential retention of urodelefeatures in anural embryos. The second step may havebeen abbreviation of some of the larval differentiationpathways. As described above, examples of abbrevi-ated programs exist in the notochord, primary muscle,and sensory cell lineages of extant anural species. Thethird step may have been elimination of larvalprograms, which has occurred in the primary andsecondary muscle lineages and brain sensory celllineage in many anural species. Finally, heterochronicacceleration in development of the larval tunic(38) andjuvenile respiratory appendages(2e'32'47) observed insome anural species would also be due to dissociability,

BioEssays Vol, 14, No. 4 - April 1992 22g

and therefore are an effect rather than a cause of anuraldevelopment.

Interspecific Hybridization Shows That Maternaland Zygotic Processes Control AnuralDevelopmentGiven that multiple mechanisms may underlie anuraldevelopment, it is important to establish whether thepoints of departure from the urodele program occur inthe egg, the embryo, or both. This question wasaddressed by interspecific hybridization experimentswith the.closely-related species M. oculata and M.occultat"). These species have similar adult mor-phologies and cross-hybridize in the laboratory, yetexhibit radically different modes of developmenl. M.oculata is a urodele developer with a conventional larva(Figs 2A and 4C), whereas M. occulta is an anuraldeveloper which lacks a tadpole stage (Figs 28 and4A).The urodele and anural species have eggs of about thesame size and develop at a similar rate up to thehatching stage (Table L). Therefore, in contrast toadultation and caudalizarion, neither changes in eggsize nor developmental timing appear to play a causalrole in the evolution of anural development.

The most informative experiment was.fertilization ofM. occulta eggs with M. oculata sperm(r)'. Remarkably,some of the hybrid embryos produced by this crossdeveloped a brain pigment cell and a short tailcontaining an extended notochord (Fig. 48). Inaddition, the urodele complement of AChE-positivemuscle lt^qgug" cells was expressed in these hybridembryos("). The development of these urodele featuresis strictly dependent on the paternal (urodele) genome,rather than another component of the M. oculata sperm(such as the centrosome), because gynogenetic hybrids,produced by fertilizing M. occulta eggs with UV-irradiated M. oculata sperm, do not develop pigmentcells or tails (Jeffery and Swalla, in preparation).Instead, the gynogenetic hybrids exhibit only anuraldevelopment. The partial restoration of urodelefeatures due to zygotic expression of the urodelegenome in the anural cytoplasm suggests that thegenetic switch to anural development involves /oss offunctions. In support of this possibility, the reciprocalcross (fertilization of M. oculata eggs with M. occultasperm) leads to the development of hybrids withurodele, rather than anural, features.

Further phenotypic examination of hybrids producedby fertilizing M. occulta eggs with M. oculata spermestablished that some urodele features are not re-stored(3s). The muscle lineage cells of hybrid embryosdo not express contractile protein genes or assemblemyofilaments, and the undifferentiated muscle cells failto enter the tail with the extending notochord. Inaddition, there is no increase in the number ofnotochord lineage cells, implying that the short tail ofhybrid embryos is due to fewer differentiated noto-chord cells rather than incomplete morphogenesis.

Therefore, changes in maternal factors appear to beresponsible for lack of primary muscle cell differen-tiation and reduction of the notochord linease in anuralembryos.

The Myoplasm is Disrupted in Anural EggsAs mentioned earlier, there is evidence that muscledeterminants reside in the myoplasm of ascidianeggs(ls). The myoplasm is a unique cytoskeletal domainconsisting of a network of actin filaments localizedbeneath the plasma membrane and a lattice ofintermediate filaments concentrated deeper in the eggcortex(a). The filamentous lattice contains embeddedlipid granules, .which color the myoplasm in someascidian species(o), and mitochondria, which providethe energy for larval locomotion. The lipid granules andmitochondria are not responsible for determiningmuscle cells because they can be displaced from themyoplasm by weak..centrifugation without affectingmuscle development(*'). In contrast, stronger centrifu-gation, whiclr displaces the filamentous lattice from themyoplasm(*o', changes the location of muscle celldiifelentiation in the embryo(45). These results suggestthat the filamentous lattice of the myoplasmic cytoskel-etal domain is an important factor in muscle celldetermination.

An early indication that differences may exist in themyoplasm in urodele and anural ascidians came from aninvestigation of the distribution of the mitochondrialenzyme succinic dehydrogenase(43). The results showedthat this enzyme is localized in the myoplasm of urodelespecies but distributed throughout the cytoplasm ineggs of the anural ascidian Molgula arenata, suggestingthat the myoplasm of anural ascidian eggs may bedefective in localizing mitochondria.

More recently, the integrity of the myoplasm inanural eggs was investigated with antibodies thatrecognize ditferent myoplasmic components(47). Stain-ing with actin antibody showed that the network of actinfilaments is present and segregated normally to themuscle lineage blastomeres of most anural ascidians. Incontrast, staining with NN18, a monoclonal antibodythat recognizes a 58 kDa protein in the filamentouslattice, showed a striking difference in localization ofmyoplasm in urodele and anural eggs. Although the 58kDa antigen was concentrated in the myoplasm of everyurodele species examined, it was uniformly distributedand significantly reduced in tifg.r in eggs of phylogeneti-cally-diverse anural speciest"". In both urodele andanural species, the 58 kDa antigen first appears in theperinuclear region and then spreads to the oocytecortex during vitellogenesis, but in anural species itsubsequently disperses throughout the cytoplasm,rather than becoming localized in the myoplasm. Thisresult suggests that the organization of the myoplasmhas been altered in species with anural development. Inthe urodele ascidian Phallusia mammillata, a small partof the myoplasm segregates into the A4.1 quadrant,


where it could be inherited by presumptive notochordcells(a8). Thus, disruption of the myoplasmic cytoskel-eton could promote anural development by affectingthe segregation and function of muscle and notochorddeterminants.

Gell Interactions May Be Altered During AnuralDevelopmentAlthough little is known about the nature of zygoticchanges during anural development, evolutionarychanges in cell interactions are likely to be involved. Amodification in notochord cell intercalation, forexample, could prevent the subsequent extension of thenotochord in anural embryos. Likewise, altered cellinteractions could explain the lack of brain-sensory andsecondary muscle cell differentiaion during anuraldevelopment, because these larval prog_rams areinitiated by induction in urodele etnbryostrv-z'). Therole of inductive cell interactions could be tested bytransplanting the inducing and responding tissuesbetween anural and urodele embryos.

GonclusionsAlthough much remains to be understood about theevoltuionary processes responsible for alternate modesof development in ascidians, several important con-clusions can be made from the available information.First, adultation is the only mode of developmentwhose origin can be attributed solely to heterochrony.The primary cause of caudalization appears to be achange inlhe number of cell divisions in the tail muscleprogram. Anural development involves the abbrevi-ation or elimination of multiple developmental pro-grams. A key event in establishing different modes ofdevelopment may be dissociability because it would benecessary before developmental programs could bemanipulited by other evblutionary processes(ae). Theinability of heterochrony to completely explain changesin deve-lopment was previously recognized in seaurchins()'). Second. the alternate modes of ascidiandevelopment are dependent on maternal changesduring oogenesis, possibly including the extent of yolkdeposition, accumulation of critical cell cycle com-ponents, and reorganization of the egg cytoskeleton,and zygotic changes during cleavage and early embryo-genesis, such as reallocation of cell lineages andalteration of morphogenetic cell movements during andafter gastrulation. Significant modifications in earlydevelopmental events also have been observed in directdeveloping sea urchins(a). Clearly, the concept thatearly developmental events are more constrained thanlater events must be reconsidered. Third, anural specieshave arisen from urodele ancestors and have evolvedmultiple times during the evolution of ascidians,suggesting that the mechanism(s) underlying anuraldevelopment involves relatively few genetic changes.Fourth, anural development involves the dissociation

and independence of multiple programs of larvaldevelopment. This has resulted in vestigial expressionof ancestral urodele features in anural ascidian species.Finally, the restoration of urodele features in hybridsbetween anural and urodele ascidians implies that /ossof function mutations may have been in part responsiblefor the evolution of anural development. This brings upthe exciting possibility that aspects of urodele develop-ment may be rescued by expressing urodele controlgenes or introducing their products into eggs of anuralascidians.

Now that some of the parameters responsible foralternate developmental modes have been identified, itwill be important to investigate the molecular andcellular mechanisms that underly these evolutionarychanges. Because of their unique qualities, especiallythe existence of closely related, hybridizable specieswith alternate modes of development, ascidians offer an' attractive system to determine how developmentaldifferences may have arisen during evolution.

AcknowledgementsThis essay is dedicated to Professor N. J. Berrill whohas done pioneering work on the evolution of alternatemodes of development in ascidians. Our research onanural ascidians has been supported by NIH grants HD-13970 and HD-07493 and NSF grant 9115543 andconducted in part at Station Biologique, Roscoff,France.

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William R. Jeffery* and Billie J. Swalla are at theDepartment of Zoology and Bodega MarineLaboratory, University of California, Davis, P.O.Box 247, Bodega Bay, CA 94923,1J5A.*Author For Correspondence.


evolution of alternate modes of development in ascidians

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